Serum transferrin is the protein responsible for the transport of iron between sites of assimilation, utilization, and storage. The mechanism by which low molecular weight chelating agents remove ferric ion from this protein is still not clearly understood. Characterization of the iron removal process is of fundamental important for understanding intracellular iron release and for the development of new chelating agents for the treatment of iron overload that results from the chronic transfusion therapy associated with anemias such as -thalassemia. Conflicting mechanisms have been proposed to explain the saturation kinetics observed with catecholate, phosphate, phosphonate, and hydroxamate ligands. During the initial period of this project, it was shown that neither mechanism in its simplest form is correct, and a modified rate law was proposed which includes two parallel pathways for iron removal. The purpose of the proposed research is to delineate the mechanistic steps associated with each pathway. These studies will also determine the kinetic differences between the two transferrin binding sites. Iron removal kinetics for ferric ion will be followed using visible spectrophotometry. Rapid scanning spectrophotometry will be used to follow the donation of ferric ion to apotransferrin and the reactions of bicarbonate with ternary transferrin complexes containing a phosphonate synergistic ion. Kinetic studies on the removal of trivalent A1 and In from transferrin will be included, using the uv band at 295 nm to follow the reaction. These metal ions are respectively more inert and more labile than ferric ion, and thus one should observe shifts in reaction rates for steps that involve breaking of iron-ligand bonds. Cyclic voltammetry with chemically modified electrodes appears to distinguish two forms of ferric transferrin which differ in their rate of reduction. This technique will be used to directly monitor conformation changes of ferric transferrin as a function of pH, temperature, and added salts. High added salts. High field 31p nmr will be used to characterize the interactions of the phosphonates with both apo- and diferric transferrin. The objective is to identify the purported mixed-ligand intermediates involved in iron removal by these ligands, characterize the ligand binding site, and estimate its distance from the ferric ion.